Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector

Abstract

Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications.

Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking

“…This reflects the conditions that the AAVs encounter after oral administration. However, although successful gene expression in mice has been reported for AAV vectors after oral administration [ 36 , 37 ], the transduction results here highlight the necessity to minimize the exposure time to low-pH and high-temperature conditions to preserve long-term AAV infectivity. This includes during different AAV purification techniques, e.g., nanobody affinity chromatography [ 38 ], iodixanol gradient centrifugation [ 27 ], or low pH flocculation [ 39 ], utilizing acidic reagents that come into contact with the virus.…”

Targeting Visceral Fat by Intraperitoneal Delivery of Novel AAV Serotype Vector Restricting Off-Target Transduction in Liver

“…injected to male BALB/c mice at a dose of 2 × 10 10 vg per mouse, which is similar to the doses previously used in i.v. injection, oral administration, or direct fat pad injection 7, 12. 2 weeks post-Rec2 administration, mice were subjected to in vivo bioluminescence measurement, and strong luciferase activity was observed primarily in the abdomen (Figure 1A).…”

“…Direct injection of Rec2 vector at a moderate dose (1 × 10 9 to 1 × 10 10 vg per fat depot) is sufficient to modulate the function of the targeted fat depot (BAT or SAT) and result in systemic metabolic changes 7, 8, 9, 10. Rec2 vector is efficient for both overexpression and knockdown applications 7, 8, 9, 10, 12, 27. However, up to date, scarce studies report systemic administration of AAV to transduce multiple adipose depots.…”

Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors

“… 65 Rec2 C57BL/6, 6 weeks CBA-GFP intra-iWAT 1 × 10 10 4 weeks effective C57BL/6, 6 weeks CBA-GFP direct intraperitoneal WAT injection 1 × 10 10 10 days effective IR flox , male, 6 weeks CBA-Cre intra-BAT 1 × 10 9 8 weeks effective IR flox , female, 6 weeks CBA-Cre intra-iWAT 1 × 10 10 8 weeks effective Huang et al. 69 Rec2 C57BL/6, 6 weeks CBA-GFP oral 5 × 10 9 2 weeks effective (BAT) C57BL/6, 6 weeks CBA-GFP intra-BAT 1 × 10 9 2 weeks effective C57BL/6, 6 weeks CBA-GFP intravenous 2 × 10 10 2 weeks ineffective C57BL/6, 6 weeks CBA-GFP oral 2 × 10 10 2 weeks effective (BAT) C57BL/6, 6 weeks CBA-GFP-miR122(4)-WPRE oral 2 × 10 10 2 weeks effective (BAT) VEGF (loxP) CBA-Cre oral 2 × 10 10 6 weeks effective (BAT) Zhu et al. 70 Rec2 TRE-Cx43 CBA-Cx43 intra-iWAT 1 × 10 9 4 weeks effective <...>…”

“…Among the six natural and engineered serotypes being compared (Rec1–Rec4, AAV1, AAV8) with a dose of 2 × 10 10 vg per mouse via oral gavage, Rec2 achieved the highest GFP reporter level in BAT of adult C57BL/6 mice. 69 Decreasing the dose highly favored transduction of BAT than liver. Oral administration of Rec2-GFP at the dose of 5 × 10 9 vg per mouse resulted in GFP content in BAT at least 10-fold higher than that in the liver.…”